ASSAYING BIOLOGICAL SAMPLES

Abstract: There is disclosed method and apparatus for assaying a biological sample for pharmacologically active compounds comprising forming a potentially reactive system of the sample and a fluorophore-conjugated reagent specific to a target compound, separating reacted and unreacted reagent and detecting fluorophore fluorescence in one of the separated components, the detected fluorescence being at near infra-red wavelength.

Description: ASSAYING BIOLOGICALSAMPLES This invention relates to assaying biological samples. One particular assay which is increasingly required is of foodstuffs and an example is the assay of meat for the presence of antibiotic residues or residues of other administered, pharmacologically active substances which either should not be present or which should be present only up to prescribed limits. Numerous assay procedures have been devised and developed, based mostly on the procedures used by research technologists working in the laboratory. Very often these procedures are time-consuming and do not give a result in a time which is useful for routine testing of foodstuffs. They are, moreover, quite often highly dependent on correct performance of complex, expert procedures and this would make them too costly for routine testing to the extent contemplated in various official directives and recommendations. Some procedures have been developed in which fluorophores are used as markers. Fluorescence is stimulated and detected to indicate the presence or absence of a target substance. These technique*; are dependent upon the use of very carefully prepared samples, as some naturally occurring biological substances - blood serum, for example display considerable fluorescence which can mask the fluorophore emission and lead to false results, and this is particularly so for UV excitation. These problems militate against the use of fluorescent markers in routine testing, especially where it is desired to have a test which gives rapid results and which can be used inexpensively and on a large scale in places where food is handled - abattoirs, for instance, as compared to laboratories. The present invention provides an assay method and apparatus that overcomes the problems aforementioned. The invention comprises a method for assaying a biological sample for pharmacologically active compounds comprising forming a potentially reactive system of the sample and a fluorophore-conjugated reagent specific to a target compound, separating reacted and unreacted reagent and detecting fluorophore fluorescence in one of the separated components, the detected fluorescence being at near infra-red wavelength. The sample may be a liquid sample, which may comprise a pressate, for example, of biological tissue. The reagent may be in liquid form. After equilibrium the system may be contacted with a separator for the unreacted reagent. The reagent may comprise an antibody that binds the target compound, and unreacted reagent may then be separated by an antigen. Unreacted reagent may be captured on to a solid support. The fluorophore may be excited to fluoresce by radiation at a wavelength offset from its fluorescent wavelength. The invention also comprises apparatus for assaying a biological sample for pharmacologically active compounds comprising a reaction chamber for a potentially reactive system of a sample and a fluorophore-conjugated reagent specific to a target compound, and a separation arrangement for separating reacted and unreacted reagent after equilibration of the system and a fluorescence detector detecting fluorescenceat near infra-red wavelength in one of the separated components. The separation arrangement may comprise a travelling strip on which a separator agent is supported to which the equilibrated system is applied. The apparatus may comprise a wash to remove the system from the separation arrangement after separation has been effected. The travelling strip may support a plurality of separator agents. The apparatus may comprise an input arrangement for reagent to the reaction chamber comprising a plurality of reagent sources each capable of applying reagents specific to different target compounds and a plurality of separation arrangements each appropriate to a particular reagent and synchronising means admitting reagent to the reaction chamber and presenting the appropriate separation arrangement for application of the equilibrated system. The fluorescence detector may be adapted to detect fluorescence at a plurality of wavelengths, and may be adapted to discriminate between fluorescence at different wavelengths. The apparatus may comprise an illuminant arrangement for exciting the flurophore to fluoresce, and such illuminant arrangement may comprise a near infra-red light emitting diode. Illumination may be directed at the fluorophore via an optical fibre arrangement and likewise fluorescence may be directed at a detector via an optical fibre arrangement. Embodiments of apparatus and methods for assaying a biological sample according to the invention will now be described with reference to the accompanying drawings, in which :- Figure 1 is a general flow diagram for one method of assay; Figure 2 is a diagrammatic illustration of one embodiment of apparatus; and Figure 3 shows the results of a method according to the invention. The drawings illustrate a method for assaying a biological sample - which could be blood serum or a tissue pressate, for example, or any other biological sample as will be further discussed below - for pharmacologically active compounds. Such compounds include drugs (whether therapeutic agents or, in horses and humans, for example, drugs of abuse) and may also include residues by which the earlier presence of drugs or other administered substances may be deduced. The method comprises forming a potentially reactive system of the sample and a fluorophore- conjugated reagent specific to a target compound. The target compound may be an antibiotic, a steroid, a toxin such as a ycotoxin or a residue suspected to be present in the sample and the reagent. More specifically, for the assay of animal tissue, the target substance may be a drug such as Chloramphenicol, Streptomycin, Penicillin or a tetracycline, or may be a residue or a combination of residues of drugs such as sulphonamides and nitrofurans. The method may be applied qualitatively, to detect any presence of a target substance, perhaps, in the case of very low concentrations, after amplification, or quantitatively, to indicate a level of concentration for comparison, say, with an officially prescribed maximum permitted level. As will be further explained below, the assay may be devised to yield a "go/no-go" result which will facilitate its use in routine testing for example in abattoirs and other places where food is handled or processed. The equilibrated reactive system is then subjected to an operation to separate reacted and unreacted reagent. If, of course, the target substance is not present in the sample, none of the fluorophore- conjugated reagent will have reacted with anything. If the target substance is present, some or all of the reagent will have reacted with it. Separation can be for example by contacting the equilibrated reaction system with a separator such as an antibody for the unreacted reagent. Separation leaves two components, one containing unreacted reagent (if any) and the other only reacted reagent (if any). One or other component is then examined for the presence of the fluorophore. This can be done by exciting the fluorophore to fluoresce for example by illumination with radiation of a specific wavelength and observing any resulting fluorescence using a photosensor. Specifically, the flurophores used in this method are fluorescent in the near infra-red, and may be excited by near infra-red illumination which may be of a wavelength offset from the fluorescent emission. Simultaneous assay for different target substances may be effected by using a mix of reagents specific to one of the targets, differently tagged with fluorophores so that each will be detectable at a specific near infra-red wavelength. It is found that the use of near infra-red fluorescence, especially using near infra-red excitation, avoids the signals being masked by fluorescent compounds which occur naturally in biological fluids such as protein solutions and for example blood serum and which are excited to fluoresce at high intensities by for example UV excitation, which is commonly used. By contrast, such naturally occurring fluorescents are not excited to fluoresce by a relatively low energy excitation by near infra-red wavelengths. Hitherto, immunofluoroassay, because of the standard UV excitation, has required the very careful preparation of samples to exclude naturally-occurring fluorescent substances in order to avoid the meaningful signals being masked or washed out by the background fluorescence. This has restricted the application of fluorescent assay essentially to use in research laboratories rather than as a tool for routine field testing. Figure 2 illustrates diagrammatically an instrument which is particularly well adapted to field testing. Reagent and sample are introduced in liquid phase at inlets 21,22 to a reaction chamber 23 to form a reaction system which is reacted there to equilibrium. After equilibrium the system is applied to a separator 24 which is in the form of an antibody to which any unreacted reagent binds. The separator 24 is carried on a strip 25 e.g. of plastic. Such a strip supplied on a spool 26 can carry many areas of antibody 24 for carrying out successive tests. Several such areas may be arrayed side by side and multiple reaction chambers correspondingly arrayed across the strip track so that tests for more than one target substance or combination of substances can be carried out simultaneously. Otherwise, different antibody (or other) separators 24 may be placed in order along the strip and appropriate reagents introduced to the reaction chamber by a synchronising arrangement (not shown) switching between different reagent sources. The strip 25 is forwarded stepwise ' on transport rollers 27 past a wash 28 where everything is washed away except the antibody separators 24 and any unreacted reagent, with conjugated fluorphore, bound thereto. This is then advanced to a detection station at which the bound fluorophore is illuminated with near infra-red wavelength illumination from a high intensity light emitting diode 29 via an optical fibre light guide 31. Other optical fibres 32 convey the fluorescent response to a detection and recording arrangement 33. With the exciting and fluorescent wavelengths offset it is possible to filter out reflection of the exciting wavelength so that excitation can be continuous and it is not necessary to rely on transient emission after the excitation has been extinguished. In an example of the method according to the invention, antibodies were raised in Soay sheep to an antigen prepared by diazotisation of sulphamethazine conjugated to human serum albumin. Diazotised sulphamethazine was coupled to diaminododecane and reacted with the bis-reactive succinimidyl ester of the near infra red fluorescing sulphoindocyanine dye Cy 5.0. Strips of mylor backed cellulose nitrate or polysulphone were reacted with a dilution of the antibody and the remaining reactive sites blocked with gelatin in phosphate buffered saline. The antibody coated strips were dipped in solutions of sulphamethazine for up to 15 minutes, then washed in running water at room temperature. The strips were then immersed in a dilution of the conjugate for up to fifteen minutes, washed in running water and the fluorescence measured via a fibre optic probe connected to a fluorescence spectrometer. The excitation wavelength used was 648 ran, the measured emission wavelength 666 n . The results are shown in Figure 3. Whilst an analog meter has been shown as the display of the apparatus, clearly any degree of sophistication or simplification can be used in the display and recording of results. For a go/no-go system, a threshold can be pre-set either electronically or otherwise and the concentration of reagent in the reaction system and/or the amount of antibody or other detector can be selected in accordance with desired detection levels. While the separator is shown in Figure 2 as comprising e.g. antibody supported on a plastic strip, it would equally well be possible to bind the antibody (or other separator substance) to latex, paper, glass or plastic beads, glass fibre, or magnetic beads, to allow for magnetic recovery from the reaction system. The reaction system has been described as comprising sample and reagent both in liquid phase, but it would of course also be possible to provide the sample in solid phase or even to assay a gas for example by bubbling through a liquid solution or suspension of reagent, or by flowing past a reagent bound to a solid which is then contacted with a liquid system separator, and many other variants can be contemplated all within the ambit of the invention.

Claims: CLAIMS 1. A method for assaying a biological sample for pharmacologically active compounds comprising forming a potentially reactive system of the sample and a fluorophore-conjugated reagent specific to a target compound, separating reacted and unreacted reagent and detecting fluorophore fluorescence in one of the separated components, the detected fluorescence being at near infra-red wavelength. 2. A method according to claim 1, in which the sample is a liquid sample. 3. A method according to claim 2, in which the liquid sample comprises a pressate from biological tissue. 4. A method according to any one of claims 1 to 3, in which the reagent is in liquid form. 5. A method according to any one of claims 1 to 4, in which after equilibration the system is contacted with a separator for the unreacted reagent. 6. A method._according to any one of claims 1 to 5, in which the reagent comprises an antibody that binds the target compound. 7. A method according to claim 6, in which unreacted reagent is separated by an antigen. 8. A method according to any one of claims 1 to 7, in which unreacted reagent is captured on to a solid support. 9. A method according to any one of claims 1 to 8, in which the fluorophore is excited to fluoresce by radiation at a wavelength offset from its fluorescent wavelength. 10. Apparatus for assaying a biological sample for pharmacologically active compounds comprising a reaction chamber for a potentially reactive system of a sample and a fluorophore-conjugated reagent specific to a target compound, and a separation arrangement for separating reacted and unreacted reagent after equilibration of the system and a fluorescence detector detecting fluorescenceat near infra-red wavelength in one of the separated compounds. 11. Apparatus according to claim 10, in which the separation arrangement comprises a travelling strip on which a separator agent is supported to which the equilibrated sys-tem is applied. 12. Apparatus according to claim 11, comprising a wash to remove the system from the separation arrangement after separation has been effected. 13. Apparatus according to claim 11 or claim 12, in which the travelling strip supports a plurality of separator agents. 14. Apparatus according to any one of claims 10 to 13, comprising an input arrangement for reagent to the reaction chamber comprising a plurality of reagent sources each capable of supplying reagents specific to different target compounds and a plurality of separation arrangements each appropriate to a particular reagent and synchronising means admitting reagent to the reaction chamber and presenting the appropriate separation arrangement for application to the equilibrated system. 15. Apparatus according to any one of claims 10 to 14, in which the fluorescence detector is adapted to detect fluorescence at a plurality of wavelengths. 16. Apparatus according to claim 15, in which the fluorescence detector is adapted to discriminate between fluorescence at .different wavelengths. 17. Apparatus according to any one of claims 10 to 16, comprising an illuminant arrangement for exciting the fluorophore to fluoresce. 18. Apparatus according to claim 17, in which the illuminant arrangement comprises a near infra red light emitting diode. 19. Apparatus according to claim 17 or claim 18, in which illumination is directed at the fluorophore via an optical fibre arrangement. 20. Apparatus according to any one of claims 10 to 19, in which fluorescence is directed at a detector via an optical fibre arrangement.

Inventors: BLACKMORE DAVID JOHN, BLACKMORE, DAVID, JOHN
Applicants: BLACKMORE DAVID JOHN, CAMBRIDGE VETERINARY SCIENCES, BLACKMORE, DAVID, JOHN, CAMBRIDGE VETERINARY SCIENCES LIMITED
Application year: 1994
Main classification: G01N33
IPC codes: G01N33